Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

Abstract

Adoptive cell transfer (ACT) is an important approach for basic research and emerges as an effective treatment for various diseases including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. Here, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms (SNP) in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology directed repair (HDR), systematically optimize experimental parameters and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Both repairing the cause of the scurfy syndrome, a 2bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.

Drug target validation in primary human natural killer cells using CRISPR RNP

“…24 In addition, viral delivery and expression of gene-editing machinery require the target cells to be cultured for several days before the desired gene is edited, potentially losing the naïve phenotype of the target cell. A recent study showed that simply delivering plasmids that encode the gene editing machinery could efficiently target genes in T cells, 32 however, our data suggest that even highly activated NK cells are inefficient at transcribing the required genes from large plasmid templates to result in Cas9-mediated editing. It is worth noting that pMAX-GFP vector uses a CMV promoter and at 3486 bp is considerably smaller than the Px458 (9300 bp), which uses a Cbh promoter.…”

“…A recent report demonstrated that T cells could be efficiently gene‐edited by simply introducing plasmids that contain Cas9 and the gRNA (transient transfection), 32 and if applicable to NK cells, would represent the most rapid and cost‐effective method of gene‐editing. We therefore cloned one of our validated guides against human CD45 (guide #61) into the Px458 "all‐in‐one" vector 29 that encodes Cas9 gene, eGFP, and the sgRNA.…”

Molecular Design, Optimization, and Genomic Integration of Chimeric B Cell Receptors in Murine B Cells

“…For this purpose, we transfected 10 6 primary B cells with 2 μg plasmid DNA encoding for a CMV promoter-driven GFP reporter gene directly after isolation from a mouse spleen or following pre-activation on 40 LB feeder cells for 1, 2, 3, or 4 days and analyzed GFP expression by flow cytometry 24 h after transfection. We observed enhanced transfection efficiencies and viability after pre-activation compared to transfection of freshly isolated B cells, consistent with previous findings in primary T cells (49). The highest transfection efficiency was observed after 1 day of pre-activation, followed by 4 days of pre-activation, suggesting a correlation with the time points of high proliferation rates.…”

“…We observed robust transfection efficiencies (electroporation by nucleofection) in primary B cells following pre-activation and expansion on fibroblast feeder cells expressing BAFF and CD40 ligand. Cas9-RNP-mediated HDR of double-stranded DNA occurred consistently, but with relatively low efficiencies when compared to other primary lymphocyte cells, such as T cells (49, 59). Furthermore, in contrast to observations in multiple other cell types including T and stem cells, AAV delivery of the HDR donor only marginally increased HDR frequencies in primary B cells, suggesting that low HDR efficiencies are independent of template format and transfection efficiencies (52, 58).…”

MiR-23~27~24–mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation

“…S10B, we could observe reduced frequencies of SMARTA SLAM lo CXCR5 + T FH cells or BCL6 + CXCR5 + GC-T FH cells in mice receiving SMARTA CD4 + T cells retrovirally transduced with shTOX-expressing vector compared to the ones with pMDH control vector. Moreover, when Tox was disrupted through using a recently established plasmid-based RNA-guided CRISPR system ( 28 ), further decreases in both T FH and GC-T FH cells could be observed (Fig. 5, A and B).…”